IdeS, an IgG-antibody degrading enzyme produced by Streptococcus pyogenes bacteria, is a promising therapeutic candidate for treating IgG-mediated autoimmune disorders and preventing organ transplant rejection. The therapeutic use of IdeS in humans is limited by pre-existing immunity against S. pyogenes and IdeS. The proposed research will structurally characterize the IdeS-IgG complex to gain mechanistic insight into IdeS?s function and how it achieves such high specificity for IgG. Structural information gleaned from this work will be leveraged to guide resurfacing of IdeS with polyethylene glycol (PEG) chains to reduce immunogenicity and improve pharmacokinetic properties while maintaining IgG binding and cleavage activity. Specific Aim 1: Determine the molecular composition of the IdeS?IgG complex. We will use size exclusion chromatography with multi-angle static light scattering (SEC-MALS) and negative stain electron microscopy (EM) to test the hypothesis that IdeS binds as a dimer to IgG. SEC-MALS will yield the molecular weight of the IdeS-IgG complex from which we can determine the stoichiometric composition of the complex. We will use negative-stain EM to visualize this complex to characterize its overall conformation and composition. The results will help to determine whether IdeS binds IgG in a 2:1 or 1:1 ratio. Specific Aim 2: Structurally characterize the IdeS-IgG complex. We will use hydrogen-deuterium exchange (HDX) coupled to mass spectrometry and X-ray crystallography to elucidate the structure of the IdeS-IgG complex and test the hypothesis that IdeS binds to an exosite in IgG. We will identify residues in IdeS and IgG that participate in their interaction and inform on how IdeS is capable of extreme specificity for IgG. Specific Aim 3: Resurface IdeS and test in vivo. We will test the hypothesis that resurfacing IdeS with PEG (i.g., PEGylation) will enhance its therapeutic potential by reducing immunogenicity and increasing its circulatory half-life. We will use structural data from Aim 2 to guide mutation of Lys residues in IdeS necessary for IgG binding or close to binding interfaces to prevent PEGylation. Mutations will be chosen by computational analysis of the residues using Rosetta and then determining whether these mutations are evolutionarily represented using sequence homology to related enzymes. We propose that this rational approach will yield IdeS resurfaced with PEG that maintains its enzymatic activity. A real-time FP enzyme assay will be developed to validate activity of non-PEGylated and PEGylated IdeS variants. Sandwich ELISAs using rabbit polyclonal IdeS antibodies will test the ability of PEGylation to block immunogenicity in vitro. In vivo cleavage activity and clearance of a PEGylated IdeS candidate construct will be assayed in mice by measuring changes in IgG serum levels over time.